In recent years, an electron microscope such as a Scanning-Electron-Microscopy (hereinafter, referred to as SEM), a Transmission-Electron-Microscopy (hereinafter, referred to as TEM), a Scanning-Transmission-Electron-Microscopy (hereinafter, also referred to as STEM), or the like has been used for observation and analysis of samples with a fine tissue structure.
Currently, for observation of biological samples using the electron microscope, samples fixed by pre-process are used. For fixation of the sample, there are a chemical fixing method using a reducing agent such as glutaraldehyde as a fixing solution and a physical fixing method of stopping movement instantaneously by rapid freezing and retaining a structure at that time.
In the chemical fixing method, since tissue is dyed with heavy metal, it is possible to observe a structure with clear contrast, but protein contained in the sample may be destroyed, and it is impossible to capture the original structure of the biological tissue. Although the physical fixing method requires a special freezing device, artifacts are not formed by a staining agent or the like, and the original structure of the biological tissue can be retained.
An ice embedding method is a method of rapidly freezing a sample and observing the sample with the electron microscope at an extremely low temperature without fixing or dyeing with a chemical reagent. Thin film samples such as organisms, foods, polymers, or the like containing water in their tissues are introduced into liquefied ethane cooled with liquid nitrogen, are rapidly frozen at a cooling rate of 104° C./sec or more, and are observed while being embedded in amorphous ice.
PTL 1 discloses a technique of rapidly freezing and observing TEM in order to accurately observe the original tissue structure of a liquid sample such as food. In addition, NPL 1 describes TEM observation of a sample using the ice embedding method. TEM or STEM described in PTL 1 and NPL 1 uses high-acceleration voltage with acceleration voltage of 80 kV to 300 kV. On the other hand, recently, an STEM detector has been attached to the SEM with acceleration voltage of 30 kV as maximum acceleration voltage, and it has been also possible to observe a transmission image at 30 kV.